Insect pests cost the general public billions of dollars annually in losses. These losses include the expense of controlling insect pests as well as crop loss and property damage caused by the pests. Specifically ants comprise 5% of the world's hundred worst invasive alien species as reported in Lowe S., Browne M., Boudjelas S., De Poorter M. (2000) 100 of the World's Worst Invasive Alien Species A selection from the Global Invasive Species Database. Published by The Invasive Species Specialist Group (ISSG) a specialist group of the Species Survival Commission (SSC) of the World Conservation Union (IUCN), 12 pp. First published as special lift-out in Aliens 12, Dec. 2000 and electronically available at http://www.issg.org/database/species/search.asp?st=100ss. Of the 17 land invertebrates listed, 28% are ants, including fire ants (Solenopsis spp.), Argentine ant (Linepithema humile), the little fire ant (Wasmannia auropunctata), and the crazy ant (Paratrechina spp). All of these ants have substantial economic impact. More specifically, the well-documented fire ant currently infests over 320 million acres in the United States and over $6 billion per year is spent for control and damage repair (as reported in Lard, C. F., J. Schmidt, B. Morris, L. Estes, C. Ryan, and D. Bergquist. 2006. “An economic impact of imported fire ants in the United States of America.” Texas A&M University, College Station, Texas. Available online at http://fireantecon.tamu.edu). The economic sectors affected include: residential households, electric and communication systems, agriculture (crops and livestock), golf courses, commercial businesses, schools and medical facilities, and parks and recreational areas.
The red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), was introduced from Brazil into United States in the 1930's and have been found in many southern and western parts of the United States from Maryland to southern California. The red imported fire ant has become a major agricultural and urban pest throughout those parts of the United States as S. invicta can cause significant damage to soybean, citrus, corn, okra, bean, cabbage, cucumber, eggplant, potato, sweet potato, peanut, sorghum, cotton and sunflower. Their mound-building activity can damage plant roots, leading to crop loss as well as interference with mechanical cultivation of crops.
Chemical pesticides are the primary tools used to combat these insect pests. However, use of traditional chemical pesticides has disadvantages, including non-target effects on neutral or beneficial insects, as well as other animals. Chemical pesticide usage also can lead to chemical residue run-off into streams and seepage into water supplies resulting in ecosystem/environment damage. In addition, animals higher in the food chain are at risk when they consume pesticide contaminated crops or insects. The handling and application of chemical pesticides also presents exposure danger to the public and professionals, and could lead to accidental dispersal into unintended environmentally sensitive areas. In addition, prolonged chemical pesticide application may result in an insect population becoming resistance to a chemical pesticide. In order to control a traditionally chemical resistant-pest, new more potent chemical pesticides must be utilized, which in turn will lead to another resistance cycle. As such, there is a need in the art to control pest populations without the disadvantages of traditional chemical pesticides.
An approach to decrease dependence on chemical pesticides is by causing a specific gene(s) of the target-pest to malfunction by either over expression or silencing gene expression. The silencing approach utilizes RNA interference pathways to knockdown the gene of interest via double-stranded RNA. Double strand RNA (dsRNA) induces sequence—specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). RNAi is a post-transcriptional, highly conserved process in eukaryotes that leads to specific gene silencing through degradation of the target mRNA. The silencing mechanism is mediated by dsRNA that is homologous in sequence to the gene of interest. The dsRNA is processed into small interfering RNA (siRNA) by an endogenous enzyme called DICER inside the target pest, and the siRNAs are then incorporated into a multi-component RNA-induced silencing complex (RISC), which finds and cleaves the target mRNA. The dsRNA inhibits expression of at least one gene within the target, which exerts a deleterious effect upon the target.
Fire, et al. (U.S. Pat. No. 6,506,559) discloses a process of introducing RNA into a living cell to inhibit gene expression of a target gene in that cell. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. Specifically, Fire, et al. (U.S. Pat. No. 6,506,559) discloses a method to inhibit expression of a target gene in a cell, the method comprising introduction of a double-stranded ribonucleic acid into the cell in an amount sufficient to inhibit expression of the target gene, wherein the RNA is a double-stranded molecule with a first ribonucleic acid strand consisting essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second ribonucleic acid strand consisting essentially of a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene. Furthermore, the first and the second ribonucleotide strands are separately complementary strands that hybridize to each other to form the said double-stranded construct, and the double-stranded construct inhibits expression of the target gene.
In using dsRNA in controlling a target insect, one method is to engineer a baculovirus to produce a dsRNA construct in vivo as disclosed in Liu, et al. (U.S. Pat. No. 6,846,482). Salient to Liu is contacting an insect with a recombinant baculovirus wherein a first ribonucleic acid sequence corresponds to at least a portion of at least one gene endogenous to the insect to control the insect. Given the advances made in the field of transfection efficiency and RNA interference, there is a need in the art to utilize RNA interference technology without using a baculovirus as a vector. Such a method would mediate control of a target-pest without depending on variables associated with a baculovirus, such as expression and transfection of dsRNA by the baculovirus.
To utilize RNA interference as a method to regulate gene expression for control, a specific essential gene needs to be targeted. Genes associated with neurohormones represent novel potential targets. One neurohoromone gene family is the pheromone-biosynthesis-activating neuropeptide (PBAN)/pyrokinin gene family. The PBAN/pyrokinin gene produces multiple peptides, each of which are defined by a similar 5-amino-acid C-terminal sequence (FXPRLamide) that is the active core fragment for these peptides as reported in Raina, A. K. and T. G. Kempe (1992). “Structure activity studies of PBAN of Helicoverpa zea (Lepidoptera: Noctuidae).” Insect Biochem Mol Biol 22 (3): 221-225. It was subsequently determined that the five C-terminal amino acids, FXPRLamide, represented the minimal sequence required for activity as reported in Raina, A. K. and T. G. Kempe (1992) id.; Fonagy, A., L. Schoofs, et al. (1992). “Functional cross-reactivities of some locusta myotropins and Bombyx pheromone biosynthesis activating neuropeptide.” J Insect Physiol 38 (9): 651-657; Kuniyoshi, H., H. Nagasawa, et al. (1992). “Cross-activity between pheromone biosynthesis activating neuropeptide (PBAN) and myotropic pyrokinin insect peptides.” Biosci Biotechnol Biochem 56 (1): 167-8; and Raina, A. K. and T. G. Kempe (1990). “A pentapeptide of the C-terminal sequence of PBAN with pheromonotropic activity.” Insect Biochem 20 (8): 849-851.
Members of the pheromone-biosynthesis-activating neuropeptide (PBAN)/pyrokinin peptide family have been shown to have a variety of functions in insects includes: 1) stimulate pheromone biosynthesis in female moths (Raina et al., 1989); 2) induce melanization in moth larvae (Matsumoto et al., 1990; Altstein et al., 1996); 3) induce embryonic diapause and seasonal polyphenism in moths (Suwan et al., 1994; Uehara et al., 2011); 4) stimulate visceral muscle contraction (Nachman et al., 1986; Predel and Nachman, 2001); 5) accelerate puparium formation in several flies (Zdarek et al., 1997; Verleyen et al., 2004); 6) terminate pupal diapause in heliothine moths (Sun et al., 2003; Xu and Denlinger, 2003).
To date, over 200 PBAN/pyrokinin family peptides including peptides deduced from 40 species PBAN/pyrokinin genes have been identified. While it is one of the largest neuropeptide families in insects, the physiological functions of the PBAN/Pyrokinin peptides are only partially known. As such there is a need in the art to investigative whether the PBAN/Pyrokinin pathway can be used to interfere with essential developmental and/or reproductive functions of the targeted insect pests and result in abnormal development and/or lack of reproduction.
Furthermore there is a need for novel control methods that would interfere with essential developmental and/or reproductive functions of species that do not have the undesirable characteristics of traditional chemical pesticides. To that end, there is a need to develop dsRNA constructs that are engineered to interfere with essential developmental and/or reproductive functions of specific pest insects that would overcome some of the disadvantages of using traditional pesticides.